1. Field of the Invention
The invention relates broadly to homogeneous immunoassay methods for measuring an antigen, antibody or hapten analyte in a fluid sample by measuring light scatter signals from monodisperse particles in a flow particle analyzer. More particularly, the invention relates to measuring changes in light scatter signals from binding molecule-coated monodisperse polymeric microspheres as the result of the analyte-mediated binding to such microspheres of binding molecule-coated polydisperse colloidal metal particles.
2. Description of the Prior Art
Antibodies, antigens and many haptens exhibit high affinities, not only for their complementary proteins, but also for certain solid surfaces such as those found in the wells of plastic microtiter plates, walls of plastic test tubes, polymeric microspheres, and colloidal metal particles. Exploitation of these properties has led to a revolution in the field of diagnostic assay methods for the aforementioned analytes in fluid samples such as serum.
The ability to carry out antigen-antibody interactions on solid supports has greatly simplified the separation of analyte-containing immunocomplexes from unused reactants and interfering substances, such as those often present in biological fluids. Such systems are generally referred to as "solid phase immunoassays" or "immunosorbent assays", and fall within the genus of "heterogeneous immunoassays." While the phase separation steps in heterogeneous immunoassays are valuable in reducing interferences by nonspecific binding substances that generally have an adverse effect on the sensitivity of the assay method, such assays are cumbersome and expensive, and are a focal point for reliability problems in automated systems.
Additionally, such heterogeneous systems have the additional disadvantage of requiring that one or another member of the immunocomplex be labeled with a molecule that can be easily quantified. Such molecules are generally referred to as "reporter molecules" and include radioisotopes (radioimmunoassays, RIA), enzymes (enzyme-linked immunoassays generally coupled with a chromophore, ELISA), fluorescent molecules (fluorescence immunoassays, FIA), chemiluminescent molecules (CIA), gold particles, photosensitive molecules, and the like. For a review, see Kemeny, D. M., et al., Immunology Today, 7: 67 (1986). Further, because of the limited number of chromophores and fluorophores available as receptor molecules, and the extensive overlap of emission spectra of such molecules, simultaneous assay of multiple analytes are not suitable using these reporters. For example, Cambridge Biotech's simultaneous EIA assays for C. difficile Toxin A and Toxin B, HTLV-I and HTLV-II, and HIV-1 and HIV-2, are not separable. The present invention greatly simplifies separability of signals from multiple analytes in a single reaction mixture.
"Homogeneous immunoassay" is the term applied to immunoassays in which no phase separation occurs. Such systems, which include binding protein-coated particle agglutination assays, are useful because they have fewer steps to automate, and automation is mechanically, fluidically and electrically simple. Examples of immunoassays requiring no phase separation steps include: latex microsphere agglutination, hemagglutination, and fluorescence depolarization assays. Examples of latex bead agglutination assays for single analytes are found in U.S. Pat. Nos. 4,521,521, 4,184,849, 4,279,617, 4,191,739 and 4,851,329, and for multiple analytes in a single fluid sample in Hansen, U.S. Pat. No. 5,286,452. Nephelometric or turbidimetric automated systems for agglutination or fluorescence depolarization assays are simple, inexpensive to construct, and, unlike heterogeneous assays, do not require frequent maintenance of the complicated phase separation apparatus.
The presence of interfering substances in body fluids has, however, inhibited otherwise promising homogeneous immunoassay approaches from meeting the high sensitivity requirements of many medically important tests, such as are met by ELISA and RIA. For reviews of this problem see, for example, Masson et al., Methods in Enzymology, 74: 115(1981) and Collett-Casssart et al., Clin Chem., 27:64 (1981). One important aspect of the present invention is that it is a homogeneous immunoassay that is free from non-specific interferences, at least to a sensitivity level of about 5.times.10.sup.-13 M. This level of sensitivity is two to three orders of magnitude greater than prior art homogeneous latex bead agglutination assays. See, e.g., alpha-fetoprotein (3.times.10.sup.-10 M, Collett-Cassart et al. above), urinary HCG (6.times.10.sup.-11 M, Lentrichia et al., J. Immunol. Meth., 89:657(1986) (but exhibiting only an 87% correlation with RIA at analyte levels 20 times the claimed sensitivity limit), and serum digoxin by a fluorescence depolarization method (3.times.10.sup.-10 M, S. Wong in D. Chan, ed., Immunoassay Automation, Academic Press, 1992, p. 329).
Prior art approaches to eliminating or decreasing the undesirable effects of non-specific interfering substance on homogeneous immunoassays have been generally unsatisfactory. These include: high dilution of body fluid sample (Fritz et al., J. Immunol., 108:110(1972), but this proportionally decreases sensitivity; using antibody fragments (Masson, Id.), but this approach is expensive and unpredictable; and, use of special conditions of pH, ionic strength, and buffer type, and/or addition of chelators or other scavengers (Masson, Id.), but these introduce multiple dependent factors that must be optimized for each analyte, and can become prohibitively expensive and cumbersome (Lim et al., J. Clin. Chem. Clin. Biochem., 20:141(1982).
Other approaches to solving the non-specific interference problem have included using IgG-coated latex ultramicrospheres to inhibit non-specific reactions in a latex sphere agglutination assay that uses antibody fragments. The sensitivity of one such agglutination method using a Coulter principle electronic resistance flow particle analyzer with a 30 .mu.m orifice was reported to be about 5.times.10.sup.-13 to 4.times.10.sup.-12 M. Sakai et al., Chem. Pharm. Bull., 37:3010 (1989). The disadvantages of this approach is that the additional reagent (non-specific, IgG coated ultramicrospheres) has an incremental manufacturing, quality control and storage cost associated with it. The present invention removes this important disadvantage by combining the action of specific immunoreactivity with the action of improving specificity, all in one reagent. In addition, although the Coulter principle particle counter used by Sakai et al. yields quantitative results, the need for the small (30 .mu.m) orifice in order to sense agglutination has the well known problem of clogging during agglutination reactions (Masson, Id.). The present invention uses a sheath flow particle analyzer with a 250-300 .mu.m orifice, which eliminates clogging. However, in the Sakai et al. approach, the distribution of specifically agglutinated particles (dimers, trimers, etc) presents a problem in Coulter volume overlap if simultaneous assays of more than one analyte are attempted, a problem not encountered in the present invention as multimers are not formed and multiple simultaneous assays can be performed without algorithms to remove the problem of overlap.
Another problem encountered in prior art agglutination immunoassays is the need for agitation by mechanical mixers during the entire reaction period of reaction mixtures containing particles of one micron or greater (Masson, Id.), thus requiring stringent washing between samples to prevent carryover of samples. A further advantage of the present invention, insofar as automation is concerned, is that agitation of samples is not needed to complete the agglutination reaction during useful time frames.
Schutt et al., EP 0254430 and U.S. Pat. No. 5,017,009, show a scattered total internal reflectance (STIR) assay method for an analyte in which colloidal gold particles are used as a label for proteins that bind to a coated macroscopic plastic plate of optical quality. This immunoassay relies upon the detection of back scattered light from an evanescent wave disturbed by the presence of a colloidal gold label brought to the interface by an immunological reaction. This evanescent wave is said to be the result of a totally internally reflected incident light wave. A disadvantage of this system is that the expensive optical-quality plastic plate is not reusable and must be discarded after a single use.
There remains an important need for an immunoassay method for antigens, antibodies, and haptens in fluid samples that combines the mechanical simplicity and low cost of particle agglutination homogeneous assays with the reduction in deleterious effects of interfering substances enjoyed by solid support based heterogeneous assays, and that does so with increased efficiency and scope when compared to prior art agglutination assays. This need is now fulfilled by the invention described in detail below.